scholarly journals Overcoming tissue scattering in wide-field deep imaging by extended detection and computational reconstruction

2019 ◽  
Author(s):  
Yuanlong Zhang ◽  
Tiankuang Zhou ◽  
Xuemei Hu ◽  
Hao Xie ◽  
Lu Fang ◽  
...  
Keyword(s):  
Multiphoton Microscopy ◽  
Tissue Imaging ◽  
Wide Field ◽  
Temporal Focusing ◽  
Tissue Scattering ◽  
Deep Imaging ◽  
Detection Mode ◽  
Computational Reconstruction ◽  
Imaging Depth

AbstractCompared to the golden technique of point‐scanning multiphoton microscopy, line‐scanning temporal focusing microscopy (LTFM) is competitive in high imaging speed while maintaining tight axial confinement. However, considering its wide‐field detection mode, LTFM suffers from shallow penetration depth as a result of crosstalk induced by tissue scattering. In contrast to the spatial filtering based on confocal slit detection, we propose the extended detection LTFM (ED‐LTFM), the first technique to extract signals from scattered photons and thus effectively extend the imaging depth. By recording a succession of line‐shape excited signals in 2D and reconstructing signals under Hessian regularization, we can push the depth limitation in scattering tissue imaging. We valid the concept with numerical simulations, and demonstrate the performance of enhanced imaging depth in in vivo imaging of mouse brains.

scholarly journals De-scattering with Excitation Patterning enables rapid wide-field imaging through scattering media

2021 ◽  
Vol 7 (28) ◽  
pp. eaay5496
Author(s):  
Cheng Zheng ◽  
Jong Kang Park ◽  
Murat Yildirim ◽  
Josiah R. Boivin ◽  
Yi Xue ◽  
...  
Keyword(s):  
High Resolution ◽  
Structural Features ◽  
Computational Imaging ◽  
Tissue Imaging ◽  
Wide Field ◽  
Scattering Media ◽  
Temporal Focusing ◽  
Field Imaging ◽  
Wide Field Imaging

Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A key unmet challenge is its limited throughput especially compared to rapid wide-field modalities that are used ubiquitously in thin specimens. Wide-field imaging methods in tissue specimens have found successes in optically cleared tissues and at shallower depths, but the scattering of emission photons in thick turbid samples severely degrades image quality at the camera. To address this challenge, we introduce a novel technique called De-scattering with Excitation Patterning or “DEEP,” which uses patterned nonlinear excitation followed by computational imaging–assisted wide-field detection. Multiphoton temporal focusing allows high-resolution excitation patterns to be projected deep inside specimen at multiple scattering lengths due to the use of long wavelength light. Computational reconstruction allows high-resolution structural features to be reconstructed from tens to hundreds of DEEP images instead of millions of point-scanning measurements.

scholarly journals Single-cell analysis of circadian dynamics in tissue explants

2015 ◽  
Vol 26 (22) ◽  
pp. 3940-3945 ◽  
Author(s):  
Laura Lande-Diner ◽  
Jacob Stewart-Ornstein ◽  
Charles J. Weitz ◽  
Galit Lahav
Keyword(s):  
Circadian Clock ◽  
Single Cell ◽  
Cell Tracking ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Tissue Imaging ◽  
Wide Field ◽  
Isolated Cells ◽  
Peripheral Tissues

Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models.

scholarly journals In vivo multiphoton fluorescence imaging with polymer dots

2017 ◽  
Author(s):  
Ahmed M. Hassan ◽  
Xu Wu ◽  
Jeremy W. Jarrett ◽  
Shihan Xu ◽  
David R. Miller ◽  
...  
Keyword(s):  
Multiphoton Microscopy ◽  
Multiphoton Imaging ◽  
Two Photon ◽  
Photon Excitation ◽  
Deep Imaging ◽  
Polymer Dots ◽  
Versatile Tool ◽  
Cortical Imaging ◽  
Semiconducting Polymer Dots

AbstractDeep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two-versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 – 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 μm beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths (λex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 μm and 1300 μm. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.

scholarly journals HiLo Based Line Scanning Temporal Focusing Microscopy for High-Speed, Deep Tissue Imaging

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 634
Author(s):  
Ruheng Shi ◽  
Yuanlong Zhang ◽  
Tiankuang Zhou ◽  
Lingjie Kong
Keyword(s):  
High Speed ◽  
Three Dimensions ◽  
Tissue Imaging ◽  
Deep Penetration ◽  
High Temporal Resolution ◽  
Structured Illumination ◽  
Volumetric Imaging ◽  
Temporal Focusing ◽  
Line Scanning

High-speed, optical-sectioning imaging is highly desired in biomedical studies, as most bio-structures and bio-dynamics are in three-dimensions. Compared to point-scanning techniques, line scanning temporal focusing microscopy (LSTFM) is a promising method that can achieve high temporal resolution while maintaining a deep penetration depth. However, the contrast and axial confinement would still be deteriorated in scattering tissue imaging. Here, we propose a HiLo-based LSTFM, utilizing structured illumination to inhibit the fluorescence background and, thus, enhance the image contrast and axial confinement in deep imaging. We demonstrate the superiority of our method by performing volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo.

Signal to Noise Considerations in Single Pixel Multiphoton Microscopy

2020 ◽  
Vol 5 (5) ◽  
pp. 1-2
Author(s):  
Dushan Wadduwage
Keyword(s):  
Multiphoton Microscopy ◽  
Tissue Imaging ◽  
Wide Field ◽  
Signal To Noise ◽  
Deep Tissue ◽  
Multiplexed Imaging ◽  
Deep Tissue Imaging ◽  
Single Pixel ◽  
Noise Metrics

Single-pixel imaging geometries for wide-field multiphoton microscopy (SPx-MPM) have emerged as a contender to conventional point-scanning multiphoton systems (PS-MPM) for deep tissue imaging. These systems are thought to be faster due to their multiplexed imaging capabilities with higher photon throughput. In this study we numerically compare the signal to noise metrics of the SPx-MPM to the PS-MPM systems. Our results suggest that PS-MPM systems outperform SPx-MPM systems, despite their higher photon throughput.

scholarly journals Advanced optical microscopy methods for in vivo imaging of sub-cellular structures in thick biological tissues

2014 ◽  
Vol 07 (05) ◽  
pp. 1440001 ◽  
Author(s):  
Nanguang Chen ◽  
Shakil Rehman ◽  
Colin J. R. Sheppard
Keyword(s):  
Optical Microscopy ◽  
Multiphoton Microscopy ◽  
Biological Tissues ◽  
Cellular Structures ◽  
High Resolution Images ◽  
Novel Method ◽  
Imaging Depth ◽  
Indispensable Tool ◽  
Time Image

Optical microscopy has become an indispensable tool for visualizing sub-cellular structures and biological processes. However, scattering in biological tissues is a major obstacle that prevents high-resolution images from being obtained from deep regions of tissue. We review common techniques, such as multiphoton microscopy (MPM) and optical coherence microscopy (OCM), for diffraction limited imaging beyond an imaging depth of 0.5 mm. Novel implementations have been emerging in recent years giving higher imaging speed, deeper penetration, and better image quality. Focal modulation microscopy (FMM) is a novel method that combines confocal spatial filtering with focal modulation to reject out-of-focus background. FMM has demonstrated an imaging depth comparable to those of MPM and OCM, near-real-time image acquisition, and the capability for multiple contrast mechanisms.

scholarly journals A microrobotic system guided by photoacoustic computed tomography for targeted navigation in intestines in vivo

2019 ◽  
Vol 4 (32) ◽  
pp. eaax0613 ◽  
Author(s):  
Zhiguang Wu ◽  
Lei Li ◽  
Yiran Yang ◽  
Peng Hu ◽  
Yang Li ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Biomedical Applications ◽  
Near Infrared ◽  
Tissue Imaging ◽  
Infrared Light ◽  
Precise Control ◽  
Deep Imaging ◽  
Diverse Environment ◽  
Deep Tissue Imaging

Recently, tremendous progress in synthetic micro/nanomotors in diverse environment has been made for potential biomedical applications. However, existing micro/nanomotor platforms are inefficient for deep tissue imaging and motion control in vivo. Here, we present a photoacoustic computed tomography (PACT)–guided investigation of micromotors in intestines in vivo. The micromotors enveloped in microcapsules are stable in the stomach and exhibit efficient propulsion in various biofluids once released. The migration of micromotor capsules toward the targeted regions in intestines has been visualized by PACT in real time in vivo. Near-infrared light irradiation induces disintegration of the capsules to release the cargo-loaded micromotors. The intensive propulsion of the micromotors effectively prolongs the retention in intestines. The integration of the newly developed microrobotic system and PACT enables deep imaging and precise control of the micromotors in vivo and promises practical biomedical applications, such as drug delivery.

scholarly journals Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

2011 ◽  
Vol 16 (11) ◽  
pp. 116009 ◽  
Author(s):  
Jiun-Yann Yu ◽  
Chun-Hung Kuo ◽  
Daniel B. Holland ◽  
Yenyu Chen ◽  
Mingxing Ouyang ◽  
...  
Keyword(s):  
Multiphoton Microscopy ◽  
Tissue Imaging ◽  
Wide Field ◽  
Optical Sectioning ◽  
Live Tissue ◽  
Plane Projection
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